In analysis of biological phenomena of cells and diagnoses of disease factors, detections and diagnoses at the molecular level have been required. In order to achieve this, it is required to detect a specific protein and a specific nucleic acid sequence, and the fluorescence has been widely used for the detection. Specifically, a method using a fluorescent substance that increases the fluorescence intensity by binding to a target substance such as a target protein or a target nucleic acid sequence has been known. As the fluorescent substance, for example, a substance that exhibits a Förster (fluorescence) resonance energy transfer (FRET) effect or a substance that emits fluorescence by being irradiated with excitation light has been used.
For example, in the Molecular Beacon method described in Non-Patent Literature 1, a nucleic acid in which different respective dyes have been introduced into the 5′-end and the 3′-end of a nucleic acid sequence which independently forms a step-loop structure is used. The fluorescence is quenched by the FRET effect at the time of no hybridization, and the fluorescence is emitted when a specific hybridization occurs. This method has limitations that the sequence is required to form a stem-loop structure, and the fluorescent dyes are required to be introduced into the respective ends.
As a different quenching mechanism as a substitute for the conventional technology, a method employing an exciton effect exhibited when at least two dye molecules are aggregated in parallel has been proposed (Non-Patent Literatures 2 to 5, Patent Literature 1). This is a method using a complex labeling substance having, in the same molecule, chemical structures of at least two dye molecules that do not emit fluorescence by an exciton effect in the single strand state and emit fluorescence by resolving the aggregation state at the time when these molecules are intercalated into or groove-bound to a nucleic acid.
A primer or probe (also called an exciton oligomer) obtained by introducing this labeling substance into oligonucleotide can be used in amplification or detection of a target nucleic acid. This exciton oligomer or the like allows fluorescent switching before and after hybridization with only one type of dye, and in the case where the exciton oligomer is used for real-time monitoring of amplification reaction, it gives a sequence specific fluorescent signal. Thus, the conventional problem that non-specific amplification is also detected when an intercalator such as SYBR green I is used can be overcome. Furthermore, since a fluorophore can be introduced into dT or dC, the sequence is barely restricted.
Patent Literature 1: JP 2009-171935 A (Japanese Patent No. 4370385)
Patent Literature 2: JP 2013-183736 A
The entire subject matters of which are incorporated herein by reference.
Non-Patent Literature 1: Tyagi, S., Kramer, F. R. (1996) Nat. Biotechnol. 14, 303-308.
Non-Patent Literature 2: Ikeda S, Kubota T, Kino K, Okamoto A., Bioconjug Chem. 2008. 19. 1719-1725.
Non-Patent Literature 3: Ikeda S, Kubota T, Yuki M, Okamoto A., Angew Chem Int Ed Engl. 2009. 48. 6480-6484.
Non-Patent Literature 4: Ikeda S, Yuki M, Yanagisawa H, Okamoto A., Tetrahedron Lett. 2009, 51, 7191-7195
Non-Patent Literature 5: Takeshi Hanami, Diane Delobel, Hajime Kanamori, Yuki Tanaka, Yasumasa Kimura, Ayako Nakasone, Takahiro Soma, Yoshihide Hayashizaki, Kengo Usui, Matthias Harbers, PLOS ONE, August 2013, volume 8, Issue 8, e70942
The entire subject matters of which are incorporated herein by reference.